@article {800, title = {Multi-omic studies on missense PLG variants in families with otitis media.}, journal = {Sci Rep}, volume = {10}, year = {2020}, month = {2020 09 14}, pages = {15035}, abstract = {

Otitis media (OM), a very common disease in young children, can result in hearing loss. In order to potentially replicate previously reported associations between OM and PLG, exome and Sanger sequencing, RNA-sequencing of saliva and middle ear samples, 16S rRNA sequencing, molecular modeling, and statistical analyses including transmission disequilibrium tests (TDT) were performed in a multi-ethnic cohort of 718 families and simplex cases with OM. We identified four rare PLG variants c.112A > G (p.Lys38Glu), c.782G > A (p.Arg261His), c.1481C > T (p.Ala494Val) and c.2045~T > A (p.Ile682Asn), and one common variant c.1414G > A (p.Asp472Asn). However TDT analyses for these PLG variants did not demonstrate association with OM in 314 families. Additionally PLG expression is very low or absent in normal or diseased middle ear in mouse and human, and salivary expression and microbial α-diversity were non-significant in c.1414G > A (p.Asp472Asn) carriers. Based on molecular modeling, the novel rare variants particularly c.782G > A (p.Arg261His) and c.2045~T > A (p.Ile682Asn) were predicted to affect protein structure. Exploration of other potential disease mechanisms will help elucidate how PLG contributes to OM susceptibility in humans. Our results underline the importance of following up findings from genome-wide association through replication studies, preferably using multi-omic datasets.

}, keywords = {Animals, Ear, Middle, Female, Genomics, Humans, Male, Mice, Microbiota, Mutation, Missense, Otitis Media, Pedigree, Plasminogen, Polymorphism, Single Nucleotide, Saliva}, issn = {2045-2322}, doi = {10.1038/s41598-020-70498-w}, author = {Bootpetch, Tori C and Hafr{\'e}n, Lena and Elling, Christina L and Baschal, Erin E and Manichaikul, Ani W and Pine, Harold S and Szeremeta, Wasyl and Scholes, Melissa A and Cass, Stephen P and Larson, Eric D and Chan, Kenny H and Ishaq, Rafaqat and Prager, Jeremy D and Shaikh, Rehan S and Gubbels, Samuel P and Yousaf, Ayesha and Wine, Todd M and Bamshad, Michael J and Yoon, Patricia J and Jenkins, Herman A and Nickerson, Deborah A and Streubel, Sven-Olrik and Friedman, Norman R and Frank, Daniel N and Einarsdottir, Elisabet and Kere, Juha and Riazuddin, Saima and Daly, Kathleen A and Leal, Suzanne M and Ryan, Allen F and Mattila, Petri S and Ahmed, Zubair M and Sale, Michele M and Chonmaitree, Tasnee and Santos-Cortez, Regie Lyn P} } @article {813, title = {Otitis media susceptibility and shifts in the head and neck microbiome due to variants.}, journal = {J Med Genet}, year = {2020}, month = {2020 Jul 24}, abstract = {

BACKGROUND: Otitis media (OM) susceptibility has significant heritability; however, the role of rare variants in OM is mostly unknown. Our goal is to identify novel rare variants that confer OM susceptibility.

METHODS: We performed exome and Sanger sequencing of >1000 DNA samples from 551 multiethnic families with OM and unrelated individuals, RNA-sequencing and microbiome sequencing and analyses of swabs from the outer ear, middle ear, nasopharynx and oral cavity. We also examined protein localisation and gene expression in infected and healthy middle ear tissues.

RESULTS: A large, intermarried pedigree that includes 81 OM-affected and 53 unaffected individuals cosegregates two known rare variants, a common variant and a rare, novel pathogenic variant c.1682A>G (p.Glu561Gly) within (LOD=4.09). Carriage of the missense variant resulted in increased relative abundance of Microbacteriaceae in the middle ear, along with occurrence of Microbacteriaceae in the outer ear and oral cavity but not the nasopharynx. Eight additional novel variants were identified in 12 families and individuals with OM. A role for in OM susceptibility is further supported by lower RNA counts in variant carriers, strong SPINK5 localisation in outer ear skin, faint localisation to middle ear mucosa and eardrum and increased expression in human cholesteatoma.

CONCLUSION: variants confer susceptibility to non-syndromic OM. These variants potentially contribute to middle ear pathology through breakdown of mucosal and epithelial barriers, immunodeficiency such as poor vaccination response, alteration of head and neck microbiota and facilitation of entry of opportunistic pathogens into the middle ear.

}, issn = {1468-6244}, doi = {10.1136/jmedgenet-2020-106844}, author = {Frank, Daniel N and Giese, Arnaud P J and Hafr{\'e}n, Lena and Bootpetch, Tori C and Yarza, Talitha Karisse L and Steritz, Matthew J and Pedro, Melquiadesa and Labra, Patrick John and Daly, Kathleen A and Tantoco, Ma Leah C and Szeremeta, Wasyl and Reyes-Quintos, Maria Rina T and AHnkoob, Niaz and Llanes, Erasmo Gonzalo D V and Pine, Harold S and Yousaf, Sairah and Ir, Diana and Einarsdottir, Elisabet and de la Cruz, Rhodieleen Anne R and Lee, Nanette R and Nonato, Rachelle Marie A and Robertson, Charles E and Ong, Kimberly Mae C and Magno, Jose Pedrito M and Chiong, Alessandra Nadine E and Espiritu-Chiong, Ma Carmina and San Agustin, Maria Luz and Cruz, Teresa Luisa G and Abes, Generoso T and Bamshad, Michael J and Cutiongco-de la Paz, Eva Maria and Kere, Juha and Nickerson, Deborah A and Mohlke, Karen L and Riazuddin, Saima and Chan, Abner and Mattila, Petri S and Leal, Suzanne M and Ryan, Allen F and Ahmed, Zubair M and Chonmaitree, Tasnee and Sale, Michele M and Chiong, Charlotte M and Santos-Cortez, Regie Lyn P} } @article {699, title = {A2ML1 and otitis media: novel variants, differential expression, and relevant pathways.}, journal = {Hum Mutat}, volume = {40}, year = {2019}, month = {2019 08}, pages = {1156-1171}, abstract = {

A genetic basis for otitis media is established, however, the role of rare variants in disease etiology is largely unknown. Previously a duplication variant within A2ML1 was identified as a significant risk factor for otitis media in an indigenous Filipino population and in US children. In this report exome and Sanger sequencing was performed using DNA samples from the indigenous Filipino population, Filipino cochlear implantees, US probands, Finnish, and Pakistani families with otitis media. Sixteen novel, damaging A2ML1 variants identified in otitis media patients were rare or low-frequency in population-matched controls. In the indigenous population, both gingivitis and A2ML1 variants including the known duplication variant and the novel splice variant c.4061 + 1 G>C were independently associated with otitis media. Sequencing of salivary RNA samples from indigenous Filipinos demonstrated lower A2ML1 expression according to the carriage of A2ML1 variants. Sequencing of additional salivary RNA samples from US patients with otitis media revealed differentially expressed genes that are highly correlated with A2ML1 expression levels. In particular,~RND3 is upregulated in both A2ML1 variant carriers and high-A2ML1 expressors. These findings support a role for A2ML1 in keratinocyte differentiation within the middle ear as part of otitis media pathology and the potential application of ROCK inhibition in otitis media.

}, issn = {1098-1004}, doi = {10.1002/humu.23769}, author = {Larson, Eric D and Magno, Jose Pedrito M and Steritz, Matthew J and Llanes, Erasmo Gonzalo D V and Cardwell, Jonathan and Pedro, Melquiadesa and Roberts, Tori Bootpetch and Einarsdottir, Elisabet and Rosanes, Rose Anne Q and Greenlee, Christopher and Santos, Rachel Ann P and Yousaf, Ayesha and Streubel, Sven-Olrik and Santos, Aileen Trinidad R and Ruiz, Amanda G and Lagrana-Villagracia, Sheryl Mae and Ray, Dylan and Yarza, Talitha Karisse L and Scholes, Melissa A and Anderson, Catherine B and Acharya, Anushree and Gubbels, Samuel P and Bamshad, Michael J and Cass, Stephen P and Lee, Nanette R and Shaikh, Rehan S and Nickerson, Deborah A and Mohlke, Karen L and Prager, Jeremy D and Cruz, Teresa Luisa G and Yoon, Patricia J and Abes, Generoso T and Schwartz, David A and Chan, Abner L and Wine, Todd M and Cutiongco-de la Paz, Eva Maria and Friedman, Norman and Kechris, Katerina and Kere, Juha and Leal, Suzanne M and Yang, Ivana V and Patel, Janak A and Tantoco, Ma Leah C and Riazuddin, Saima and Chan, Kenny H and Mattila, Petri S and Reyes-Quintos, Maria Rina T and Ahmed, Zubair M and Jenkins, Herman A and Chonmaitree, Tasnee and Hafr{\'e}n, Lena and Chiong, Charlotte M and Santos-Cortez, Regie Lyn P} } @article {505, title = {FAM92A Underlies Nonsyndromic Postaxial Polydactyly in Humans and an Abnormal Limb and Digit Skeletal Phenotype in Mice.}, journal = {J Bone Miner Res}, volume = {34}, year = {2019}, month = {2019 Feb}, pages = {375-386}, abstract = {

Polydactyly is a common congenital anomaly of the hand and foot. Postaxial polydactyly (PAP) is characterized by one or more posterior or postaxial digits. In a Pakistani family with autosomal recessive nonsyndromic postaxial polydactyly type A (PAPA), we performed genomewide genotyping, linkage analysis, and exome and Sanger sequencing. Exome sequencing revealed a homozygous nonsense variant (c.478C>T, p.[Arg160*]) in the FAM92A gene within the mapped region on 8q21.13-q24.12 that segregated with the PAPA phenotype. We found that FAM92A is expressed in the developing mouse limb and E11.5 limb bud including the progress zone and the apical ectodermal ridge, where it strongly localizes at the cilia level, suggesting an important role in limb patterning. The identified variant leads to a loss of the FAM92A/Chibby1 complex that is crucial for ciliogenesis and impairs the recruitment and the colocalization of FAM92A with Chibby1 at the base of the cilia. In addition, we show that Fam92a homozygous mice also exhibit an abnormal digit morphology, including metatarsal osteomas and polysyndactyly, in addition to distinct abnormalities on the deltoid tuberosity of their humeri. In conclusion, we present a new nonsyndromic PAPA ciliopathy due to a loss-of-function variant in FAM92A. {\textcopyright} 2018 American Society for Bone and Mineral Research.

}, issn = {1523-4681}, doi = {10.1002/jbmr.3594}, author = {Schrauwen, Isabelle and Giese, Arnaud Pj and Aziz, Abdul and Lafont, David Tino and Chakchouk, Imen and Santos-Cortez, Regie Lyn P and Lee, Kwanghyuk and Acharya, Anushree and Khan, Falak Sher and Ullah, Asmat and Nickerson, Deborah A and Bamshad, Michael J and Ali, Ghazanfar and Riazuddin, Saima and Ansar, Muhammad and Ahmad, Wasim and Ahmed, Zubair M and Leal, Suzanne M} } @article {549, title = {Global genetic insight contributed by consanguineous Pakistani families segregating hearing loss.}, journal = {Hum Mutat}, volume = {40}, year = {2019}, month = {2019 Jan}, pages = {53-72}, abstract = {

Consanguineous Pakistani pedigrees segregating deafness have contributed decisively to the discovery of 31 of the 68 genes associated with nonsyndromic autosomal recessive hearing loss (HL) worldwide. In this study, we utilized genome-wide genotyping, Sanger and exome sequencing to identify 163 DNA variants in 41 previously reported HL genes segregating in 321 Pakistani families. Of these, 70 (42.9\%) variants identified in 29 genes are novel. As expected from genetic studies of disorders segregating in consanguineous families, the majority of affected individuals (94.4\%) are homozygous for HL-associated variants, with the other variants being compound heterozygotes. The five most common HL genes in the Pakistani population are SLC26A4, MYO7A, GJB2, CIB2 and HGF, respectively. Our study provides a profile of the genetic etiology of HL in Pakistani families, which will allow for the development of more efficient genetic diagnostic tools, aid in accurate genetic counseling, and guide application of future gene-based therapies. These findings are also valuable in interpreting pathogenicity of variants that are potentially associated with HL in individuals of all ancestries. The Pakistani population, and its infrastructure for studying human genetics, will continue to be valuable to gene discovery for HL and other inherited disorders.

}, issn = {1098-1004}, doi = {10.1002/humu.23666}, author = {Richard, Elodie M and Santos-Cortez, Regie Lyn P and Faridi, Rabia and Rehman, Atteeq U and Lee, Kwanghyuk and Shahzad, Mohsin and Acharya, Anushree and Khan, Asma A and Imtiaz, Ayesha and Chakchouk, Imen and Takla, Christina and Abbe, Izoduwa and Rafeeq, Maria and Liaqat, Khurram and Chaudhry, Taimur and Bamshad, Michael J and Nickerson, Deborah A and Schrauwen, Isabelle and Khan, Shaheen N and Morell, Robert J and Zafar, Saba and Ansar, Muhammad and Ahmed, Zubair M and Ahmad, Wasim and Riazuddin, Sheikh and Friedman, Thomas B and Leal, Suzanne M and Riazuddin, Saima} } @article {661, title = {FUT2 Variants Confer Susceptibility to Familial Otitis Media.}, journal = {Am J Hum Genet}, volume = {103}, year = {2018}, month = {2018 11 01}, pages = {679-690}, abstract = {

Non-secretor status due to homozygosity for the common FUT2 variant c.461G>A (p.Trp154) is associated with either risk for autoimmune diseases or protection against viral diarrhea and HIV. We determined the role of FUT2 in otitis media susceptibility by obtaining DNA samples from 609 multi-ethnic families and simplex case subjects with otitis media. Exome and Sanger sequencing, linkage analysis, and Fisher exact and transmission disequilibrium tests (TDT) were performed. The common FUT2 c.604C>T (p.Arg202) variant co-segregates with otitis media in a Filipino pedigree (LOD = 4.0). Additionally, a rare variant, c.412C>T (p.Arg138Cys), is associated with recurrent/chronic otitis media in European-American children (p = 1.2~{\texttimes} 10) and US trios (TDT p = 0.01). The c.461G>A (p.Trp154) variant was also over-transmitted in US trios (TDT p = 0.01) and was associated with shifts in middle ear microbiota composition (PERMANOVA p < 10) and increased biodiversity. When all missense and nonsense variants identified in multi-ethnic US trios with CADD > 20 were combined, FUT2 variants were over-transmitted in trios (TDT p = 0.001). Fut2 is transiently upregulated in mouse middle ear after inoculation with non-typeable Haemophilus influenzae. Four FUT2 variants-namely p.Ala104Val, p.Arg138Cys, p.Trp154, and p.Arg202-reduced A antigen in mutant-transfected COS-7 cells, while the nonsense variants also reduced FUT2 protein levels. Common and rare FUT2 variants confer susceptibility to otitis media, likely by modifying the middle ear microbiome through regulation of A antigen levels in epithelial cells. Our families demonstrate marked intra-familial genetic heterogeneity, suggesting that multiple combinations of common and rare variants plus environmental factors influence the individual otitis media phenotype as a complex trait.

}, issn = {1537-6605}, doi = {10.1016/j.ajhg.2018.09.010}, author = {Santos-Cortez, Regie Lyn P and Chiong, Charlotte M and Frank, Daniel N and Ryan, Allen F and Giese, Arnaud P J and Bootpetch Roberts, Tori and Daly, Kathleen A and Steritz, Matthew J and Szeremeta, Wasyl and Pedro, Melquiadesa and Pine, Harold and Yarza, Talitha Karisse L and Scholes, Melissa A and Llanes, Erasmo Gonzalo D V and Yousaf, Saira and Friedman, Norman and Tantoco, Ma Leah C and Wine, Todd M and Labra, Patrick John and Benoit, Jeanne and Ruiz, Amanda G and de la Cruz, Rhodieleen Anne R and Greenlee, Christopher and Yousaf, Ayesha and Cardwell, Jonathan and Nonato, Rachelle Marie A and Ray, Dylan and Ong, Kimberly Mae C and So, Edward and Robertson, Charles E and Dinwiddie, Jordyn and Lagrana-Villagracia, Sheryl Mae and Gubbels, Samuel P and Shaikh, Rehan S and Cass, Stephen P and Einarsdottir, Elisabet and Lee, Nanette R and Schwartz, David A and Gloria-Cruz, Teresa Luisa I and Bamshad, Michael J and Yang, Ivana V and Kere, Juha and Abes, Generoso T and Prager, Jeremy D and Riazuddin, Saima and Chan, Abner L and Yoon, Patricia J and Nickerson, Deborah A and Cutiongco-de la Paz, Eva Maria and Streubel, Sven-Olrik and Reyes-Quintos, Maria Rina T and Jenkins, Herman A and Mattila, Petri and Chan, Kenny H and Mohlke, Karen L and Leal, Suzanne M and Hafr{\'e}n, Lena and Chonmaitree, Tasnee and Sale, Michele M and Ahmed, Zubair M} } @article {519, title = {Peripheral Cone Dystrophy: Expanded Clinical Spectrum, Multimodal and Ultrawide-Field Imaging, and Genomic Analysis.}, journal = {J Ophthalmol}, volume = {2018}, year = {2018}, month = {2018}, pages = {2984934}, abstract = {

Purpose: To present new clinical features, multimodal and ultrawide-field imaging characteristics of peripheral cone dystrophy (PCD), and results of laboratory and genetic investigation to decipher the etiology.

Methods: Retrospective observational case-series.

Results: Three patients with PCD presented with bilateral paracentral scotomas and a mean visual acuity of 20/25. All exhibited confluent macular hyperautofluorescence with a central bull{\textquoteright}s eye lesion. Spectral-domain optical coherence tomography revealed loss of outer retinal elements, particularly the inner segment ellipsoid band and external limiting membrane, within the area of macular hyperautofluorescence. This area corresponded with a lightened fundus appearance and variable retinal pigment epithelium (RPE) abnormalities. Full field and multifocal electroretinography distinguished PCD from other photoreceptor dystrophies. Ultrawide-field imaging revealed irregular peripheral retinal lesions in a distribution greater nasally than temporally and not contiguous with the macular lesion. Functional and anatomic testing remained stable over a mean follow-up of 3 years. Laboratory investigation for causes of uveitis was negative. Whole exome sequencing identified rare variants in genes associated with macular or cone dystrophy or degeneration.

Conclusions: In contrast to the original description, the funduscopic and fluorescein angiographic appearance of PCD is abnormal, although the defects are subtle. Peripheral lesions may be observed in some patients. Bilateral, symmetric, macular hyperautofluorescence associated with outer retinal atrophy that spares the fovea is a characteristic of PCD. Pathogenic variants in the same gene were not shared across the cohort, suggesting genetic heterogeneity. Further evaluation is warranted.

}, issn = {2090-004X}, doi = {10.1155/2018/2984934}, author = {Sisk, Robert A and Hufnagel, Robert B and Laham, Ailee and Wohler, Elizabeth S and Sobreira, Nara and Ahmed, Zubair M} } @article {403, title = {Molecular outcomes, clinical consequences, and genetic diagnosis of Oculocutaneous Albinism in Pakistani population.}, journal = {Sci Rep}, volume = {7}, year = {2017}, month = {2017 03 07}, pages = {44185}, abstract = {

Nonsyndromic oculocutaneous Albinism (nsOCA) is clinically characterized by the loss of pigmentation in the skin, hair, and iris. OCA is amongst the most common causes of vision impairment in children. To date, pathogenic variants in six genes have been identified in individuals with nsOCA. Here, we determined the identities, frequencies, and clinical consequences of OCA alleles in 94 previously unreported Pakistani families. Combination of Sanger and Exome sequencing revealed 38 alleles, including 22 novel variants, segregating with nsOCA phenotype in 80 families. Variants of TYR and OCA2 genes were the most common cause of nsOCA, occurring in 43 and 30 families, respectively. Twenty-two novel variants include nine missense, four splice site, two non-sense, one insertion and six gross deletions. In vitro studies revealed retention of OCA proteins harboring novel missense alleles in the endoplasmic reticulum (ER) of transfected cells. Exon-trapping assays with constructs containing splice site alleles revealed errors in splicing. As eight alleles account for approximately 56\% (95\% CI: 46.52-65.24\%) of nsOCA cases, primarily enrolled from Punjab province of Pakistan, hierarchical strategies for variant detection would be feasible and cost-efficient genetic tests for OCA in families with similar origin. Thus, we developed Tetra-primer ARMS assays for rapid, reliable, reproducible and economical screening of most of these common alleles.

}, keywords = {Albinism, Oculocutaneous, Alleles, Female, Gene Frequency, Humans, Male, Membrane Transport Proteins, Mutation, Missense, Pakistan}, issn = {2045-2322}, doi = {10.1038/srep44185}, author = {Shahzad, Mohsin and Yousaf, Sairah and Waryah, Yar M and Gul, Hadia and Kausar, Tasleem and Tariq, Nabeela and Mahmood, Umair and Ali, Muhammad and Khan, Muzammil A and Waryah, Ali M and Shaikh, Rehan S and Riazuddin, Saima and Ahmed, Zubair M} } @article {117, title = {Identification and clinical characterization of Hermansky-Pudlak syndrome alleles in the Pakistani population.}, journal = {Pigment Cell Melanoma Res}, volume = {29}, year = {2016}, month = {2016 Mar}, pages = {231-5}, keywords = {Adolescent, Adult, Alleles, Child, Child, Preschool, Female, Hermanski-Pudlak Syndrome, Humans, Infant, Male, Membrane Proteins, Middle Aged, Pakistan, Pedigree}, issn = {1755-148X}, doi = {10.1111/pcmr.12438}, author = {Yousaf, Sairah and Shahzad, Mohsin and Kausar, Tasleem and Sheikh, Shakeel A and Tariq, Nabeela and Shabbir, Asra S and Ali, Muhammad and Waryah, Ali M and Shaikh, Rehan S and Riazuddin, Saima and Ahmed, Zubair M} } @article {42, title = {Mutational Spectrum of MYO15A and the Molecular Mechanisms of DFNB3 Human Deafness.}, journal = {Hum Mutat}, volume = {37}, year = {2016}, month = {2016 10}, pages = {991-1003}, abstract = {

Deafness in humans is a common neurosensory disorder and is genetically heterogeneous. Across diverse ethnic groups, mutations of MYO15A at the DFNB3 locus appear to be the third or fourth most common cause of autosomal-recessive, nonsyndromic deafness. In 49 of the 67 exons of MYO15A, there are currently 192 recessive mutations identified, including 14 novel mutations reported here. These mutations are distributed uniformly across MYO15A with one enigmatic exception; the alternatively spliced giant exon 2, encoding 1,233 residues, has 17 truncating mutations but no convincing deafness-causing missense mutations. MYO15A encodes three distinct isoform classes, one of which is 395 kDa (3,530 residues), the largest member of the myosin superfamily of molecular motors. Studies of Myo15 mouse models that recapitulate DFNB3 revealed two different pathogenic mechanisms of hearing loss. In the inner ear, myosin 15 is necessary both for the development and the long-term maintenance of stereocilia, mechanosensory sound-transducing organelles that extend from the apical surface of hair cells. The goal of this Mutation Update is to provide a comprehensive review of mutations and functions of MYO15A.

}, keywords = {Alternative Splicing, Animals, Deafness, Disease Models, Animal, Ear, Inner, Exons, Gene Expression Regulation, Developmental, Humans, Mice, Mutation, Myosins, Stereocilia}, issn = {1098-1004}, doi = {10.1002/humu.23042}, author = {Rehman, Atteeq U and Bird, Jonathan E and Faridi, Rabia and Shahzad, Mohsin and Shah, Sujay and Lee, Kwanghyuk and Khan, Shaheen N and Imtiaz, Ayesha and Ahmed, Zubair M and Riazuddin, Saima and Santos-Cortez, Regie Lyn P and Ahmad, Wasim and Leal, Suzanne M and Riazuddin, Sheikh and Friedman, Thomas B} } @article {204, title = {Challenges and solutions for gene identification in the presence of familial locus heterogeneity.}, journal = {Eur J Hum Genet}, volume = {23}, year = {2015}, month = {2015 Sep}, pages = {1207-15}, abstract = {

Next-generation sequencing (NGS) of exomes and genomes has accelerated the identification of genes involved in Mendelian phenotypes. However, many NGS studies fall short of identifying causal variants, with estimates for success rates as low as 25\% for uncovering the pathological variant underlying disease etiology. An important reason for such failures is familial locus heterogeneity, where within a single pedigree causal variants in two or more genes underlie Mendelian trait etiology. As examples of intra- and inter-sibship familial locus heterogeneity, we present 10 consanguineous Pakistani families segregating hearing impairment due to homozygous variants in two different hearing impairment genes and a European-American pedigree in which hearing impairment is caused by four variants in three different genes. We have identified 41 additional pedigrees with syndromic and nonsyndromic hearing impairment for which a single previously reported hearing impairment gene has been identified but only segregates with the phenotype in a subset of affected pedigree members. We estimate that locus heterogeneity occurs in 15.3\% (95\% confidence interval: 11.9\%, 19.9\%) of the families in our collection. We demonstrate novel approaches to apply linkage analysis and homozygosity mapping (for autosomal recessive consanguineous pedigrees), which can be used to detect locus heterogeneity using either NGS or SNP array data. Results from linkage analysis and homozygosity mapping can also be used to group sibships or individuals most likely to be segregating the same causal variants and thereby increase the success rate of gene identification.

}, keywords = {Asian Continental Ancestry Group, Calcium-Binding Proteins, Chromosome Mapping, Connexin 26, Connexins, Consanguinity, European Continental Ancestry Group, Female, Genes, Recessive, Genetic Heterogeneity, Genetic Linkage, Genetic Loci, Genetic Predisposition to Disease, Genome, Human, Genotype, Hearing Loss, Hepatocyte Growth Factor, High-Throughput Nucleotide Sequencing, Homozygote, Humans, Male, Membrane Transport Proteins, Mutation, Pedigree, Phenotype, Sulfate Transporters}, issn = {1476-5438}, doi = {10.1038/ejhg.2014.266}, author = {Rehman, Atteeq U and Santos-Cortez, Regie Lyn P and Drummond, Meghan C and Shahzad, Mohsin and Lee, Kwanghyuk and Morell, Robert J and Ansar, Muhammad and Jan, Abid and Wang, Xin and Aziz, Abdul and Riazuddin, Saima and Smith, Joshua D and Wang, Gao T and Ahmed, Zubair M and Gul, Khitab and Shearer, A Eliot and Smith, Richard J H and Shendure, Jay and Bamshad, Michael J and Nickerson, Deborah A and Hinnant, John and Khan, Shaheen N and Fisher, Rachel A and Ahmad, Wasim and Friderici, Karen H and Riazuddin, Sheikh and Friedman, Thomas B and Wilch, Ellen S and Leal, Suzanne M} } @article {142, title = {Identification and functional characterization of natural human melanocortin 1 receptor mutant alleles in Pakistani population.}, journal = {Pigment Cell Melanoma Res}, volume = {28}, year = {2015}, month = {2015 Nov}, pages = {730-5}, abstract = {

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor of the melanocyte{\textquoteright}s plasma membrane, is a major determinant of skin pigmentation and phototype. Upon activation by α-melanocyte stimulating hormone, MC1R triggers the cAMP cascade to stimulate eumelanogenesis. We used whole-exome sequencing to identify causative alleles in Pakistani families with skin and hair hypopigmentation. Six MC1R mutations segregated with the phenotype in seven families, including a p.Val174del in-frame deletion and a p.Tyr298* nonsense mutation, that were analyzed for function in heterologous HEK293 cells. p.Tyr298* MC1R showed no agonist-induced signaling to the cAMP or ERK pathways, nor detectable agonist binding. Conversely, signaling was comparable for p.Val174del and wild-type in HEK cells overexpressing the proteins, but binding analysis suggested impaired cell surface expression. Flow cytometry and confocal imaging studies revealed reduced plasma membrane expression of p.Val174del and p.Tyr298*. Therefore, p.Tyr298* was a total loss-of-function (LOF) allele, while p.Val174del displayed a partial LOF attribute.

}, keywords = {Alleles, Family, Female, Humans, Hypopigmentation, Male, Mutation, Pakistan, Pedigree, Phenotype, Receptor, Melanocortin, Type 1}, issn = {1755-148X}, doi = {10.1111/pcmr.12400}, author = {Shahzad, Mohsin and Sires Campos, Julia and Tariq, Nabeela and Herraiz Serrano, Cecilia and Yousaf, Rizwan and Jim{\'e}nez-Cervantes, Celia and Yousaf, Sairah and Waryah, Yar M and Dad, Haseeb A and Blue, Elizabeth M and Sobreira, Nara and L{\'o}pez-Gir{\'a}ldez, Francesc and Kausar, Tasleem and Ali, Muhammad and Waryah, Ali M and Riazuddin, Saima and Shaikh, Rehan S and Garc{\'\i}a-Borr{\'o}n, Jos{\'e} C and Ahmed, Zubair M} } @article {157, title = {Mutation of ATF6 causes autosomal recessive achromatopsia.}, journal = {Hum Genet}, volume = {134}, year = {2015}, month = {2015 Sep}, pages = {941-50}, abstract = {

Achromatopsia (ACHM) is an early-onset retinal dystrophy characterized by photophobia, nystagmus, color blindness and severely reduced visual acuity. Currently mutations in five genes CNGA3, CNGB3, GNAT2, PDE6C and PDE6H have been implicated in ACHM. We performed homozygosity mapping and linkage analysis in a consanguineous Pakistani ACHM family and mapped the locus to a 15.12-Mb region on chromosome 1q23.1-q24.3 with a maximum LOD score of 3.6. A DNA sample from an affected family member underwent exome sequencing. Within the ATF6 gene, a single-base insertion variant c.355_356dupG (p.Glu119Glyfs*8) was identified, which completely segregates with the ACHM phenotype within the family. The frameshift variant was absent in public variant databases, in 130 exomes from unrelated Pakistani individuals, and in 235 ethnically matched controls. The variant is predicted to result in a truncated protein that lacks the DNA binding and transmembrane domains and therefore affects the function of ATF6 as a transcription factor that initiates the unfolded protein response during endoplasmic reticulum (ER) stress. Immunolabeling with anti-ATF6 antibodies showed localization throughout the mouse neuronal retina, including retinal pigment epithelium, photoreceptor cells, inner nuclear layer, inner and outer plexiform layers, with a more prominent signal in retinal ganglion cells. In contrast to cytoplasmic expression of wild-type protein, in heterologous cells ATF6 protein with the p.Glu119Glyfs*8 variant is mainly confined to the nucleus. Our results imply that response to ER stress as mediated by the ATF6 pathway is essential for color vision in humans.

}, keywords = {Activating Transcription Factor 6, Adolescent, Animals, Asian Continental Ancestry Group, Color Vision Defects, Consanguinity, DNA Mutational Analysis, Exome, Female, Frameshift Mutation, Genotyping Techniques, Homozygote, Humans, Limit of Detection, Male, Mice, Mice, Inbred C57BL, Pakistan, Pedigree, Phenotype, Retina, Signal Transduction}, issn = {1432-1203}, doi = {10.1007/s00439-015-1571-4}, author = {Ansar, Muhammad and Santos-Cortez, Regie Lyn P and Saqib, Muhammad Arif Nadeem and Zulfiqar, Fareeha and Lee, Kwanghyuk and Ashraf, Naeem Mahmood and Ullah, Ehsan and Wang, Xin and Sajid, Sundus and Khan, Falak Sher and Amin-ud-Din, Muhammad and Smith, Joshua D and Shendure, Jay and Bamshad, Michael J and Nickerson, Deborah A and Hameed, Abdul and Riazuddin, Saima and Ahmed, Zubair M and Ahmad, Wasim and Leal, Suzanne M} } @article {152, title = {Rare A2ML1 variants confer susceptibility to otitis media.}, journal = {Nat Genet}, volume = {47}, year = {2015}, month = {2015 Aug}, pages = {917-20}, abstract = {

A duplication variant within the middle ear-specific gene A2ML1 cosegregates with otitis media in an indigenous Filipino pedigree (LOD score = 7.5 at reduced penetrance) and lies within a founder haplotype that is also shared by 3 otitis-prone European-American and Hispanic-American children but is absent in non-otitis-prone children and >62,000 next-generation sequences. We identified seven additional A2ML1 variants in six otitis-prone children. Collectively, our studies support a role for A2ML1 in the pathophysiology of otitis media.

}, keywords = {alpha-Macroglobulins, Animals, Base Sequence, Child, Cochlea, Exome, Family Health, Female, Gene Duplication, Gene Frequency, Genetic Predisposition to Disease, Genotype, Haplotypes, Humans, Male, Mice, Inbred C57BL, Models, Molecular, Otitis Media, Pedigree, Principal Component Analysis, Protein Conformation, Sequence Analysis, DNA}, issn = {1546-1718}, doi = {10.1038/ng.3347}, author = {Santos-Cortez, Regie Lyn P and Chiong, Charlotte M and Reyes-Quintos, Ma Rina T and Tantoco, Ma Leah C and Wang, Xin and Acharya, Anushree and Abbe, Izoduwa and Giese, Arnaud P and Smith, Joshua D and Allen, E Kaitlynn and Li, Biao and Cutiongco-de la Paz, Eva Maria and Garcia, Marieflor Cristy and Llanes, Erasmo Gonzalo D V and Labra, Patrick John and Gloria-Cruz, Teresa Luisa I and Chan, Abner L and Wang, Gao T and Daly, Kathleen A and Shendure, Jay and Bamshad, Michael J and Nickerson, Deborah A and Patel, Janak A and Riazuddin, Saima and Sale, Michele M and Chonmaitree, Tasnee and Ahmed, Zubair M and Abes, Generoso T and Leal, Suzanne M} } @article {267, title = {Adenylate cyclase 1 (ADCY1) mutations cause recessive hearing impairment in humans and defects in hair cell function and hearing in zebrafish.}, journal = {Hum Mol Genet}, volume = {23}, year = {2014}, month = {2014 Jun 15}, pages = {3289-98}, abstract = {

Cyclic AMP (cAMP) production, which is important for mechanotransduction within the inner ear, is catalyzed by adenylate cyclases (AC). However, knowledge of the role of ACs in hearing is limited. Previously, a novel autosomal recessive non-syndromic hearing impairment locus DFNB44 was mapped to chromosome 7p14.1-q11.22 in a consanguineous family from Pakistan. Through whole-exome sequencing of DNA samples from hearing-impaired family members, a nonsense mutation c.3112C>T (p.Arg1038*) within adenylate cyclase 1 (ADCY1) was identified. This stop-gained mutation segregated with hearing impairment within the family and was not identified in ethnically matched controls or within variant databases. This mutation is predicted to cause the loss of 82 amino acids from the carboxyl tail, including highly conserved residues within the catalytic domain, plus a calmodulin-stimulation defect, both of which are expected to decrease enzymatic efficiency. Individuals who are homozygous for this mutation had symmetric, mild-to-moderate mixed hearing impairment. Zebrafish adcy1b morphants had no FM1-43 dye uptake and lacked startle response, indicating hair cell dysfunction and gross hearing impairment. In the mouse, Adcy1 expression was observed throughout inner ear development and maturation. ADCY1 was localized to the cytoplasm of supporting cells and hair cells of the cochlea and vestibule and also to cochlear hair cell nuclei and stereocilia. Ex vivo studies in COS-7 cells suggest that the carboxyl tail of ADCY1 is essential for localization to actin-based microvilli. These results demonstrate that ADCY1 has an evolutionarily conserved role in hearing and that cAMP signaling is important to hair cell function within the inner ear.

}, keywords = {Adenylyl Cyclases, Animals, Cercopithecus aethiops, Codon, Nonsense, COS Cells, Cyclic AMP, Cytoplasm, Ear, Inner, Female, Hair Cells, Auditory, Hearing Loss, Humans, Labyrinth Supporting Cells, Male, Mice, Zebrafish}, issn = {1460-2083}, doi = {10.1093/hmg/ddu042}, author = {Santos-Cortez, Regie Lyn P and Lee, Kwanghyuk and Giese, Arnaud P and Ansar, Muhammad and Amin-ud-Din, Muhammad and Rehn, Kira and Wang, Xin and Aziz, Abdul and Chiu, Ilene and Hussain Ali, Raja and Smith, Joshua D and Shendure, Jay and Bamshad, Michael and Nickerson, Deborah A and Ahmed, Zubair M and Ahmad, Wasim and Riazuddin, Saima and Leal, Suzanne M} }